LU83028A1 - PHARMACEUTICAL APPROACH TO USE AS PROTECTION AGAINST ACUTE LUNG OEDEM, ANAPHYLACTIC SHOCK AND OTHER KINDS OF SHOCK AND HYPERFIBRINOLYSIS - Google Patents

Description

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Pharmaceutical approach for use as protection against acute pulmonary edema, anaphylactic shock and other types of shock as well as kyperfibrinolysis.

The invention relates to a new pharmaceutical approach which is particularly effective in acute pulmonary edema, in shock caused by bacterial endotoxins, in hydrofibrinolysis and in anaphylactic shock. In particular, the invention relates to esters of L-arginine.

Numerous esters of L-arginine are known, including, for example, esters with aliphatic alcohols, which have the general form

COOR

i · H

>. i! Θ x- Η-, Ν * NH2 * ', in which X is a pharmaceutically acceptable organic or inorganic anion and R is an alkyl group; interpret.

These esters with aliphatic alcohols are obtained by esterifying L-arginine using a method known from the literature, for example reaction of L-arginine with an aliphatic alcohol which has previously been saturated with dry, gaseous hydrogen chloride, or Esterification by removing water by azeotropic distillation or reaction with an alcohol in the presence of thionyl chloride or comparable methods. These esters, for example in the form of hydrochloric acid salts, are stable in the solid state and solutions can be prepared from the esters at neutral or slightly acidic pH. These solutions are stable over very long periods of time.

Surprisingly, it has now been found that the esters of ArgininiAt aliphatic alcohols and their salts have valuable therapeutic properties for the improvement of some pathological conditions. The material to be administered by the parenteral route consists of an aqueous solution with a pH between 5.5 and 8.5 and contains the cation of the arginine ester and an inorganic or organic anion in a molar ratio of 1: 1 or another molar ratio. In particular, the esters and their corresponding salts exert an inhibitory effect on acute pulmonary edema and increase the survival expectancy when shock symptoms occur with a variety of causes, for example because of bacterial endotoxins or because of immune endotoxin.

Furthermore, it has been shown that the L-arginine esters have valuable in vivo activity in the hyperfibrinolytic syndrome caused by administration of urokinase. It should be noted that all of these therapeutic effects are produced with doses and with the manner of administration, which do not produce any noticeable toxic effects * - 5 - * t, and that the toxicity of all these substances is very low overall. With regard to the dosage, the route of administration and overall the manner in which the effects in animals could be achieved, these substances are also useful for human therapy in the treatment of pathological conditions of the type mentioned above.

The activity of the pharmaceutical approaches according to the invention is described below on the basis of the test results.

Protection against pulmonary edema

The pulmonary edema was diagnosed using the method of A.M. Rothschild and A. Cästania (Arch. Pharmaco., 295, 177 (1976)), and substances with known therapeutic activity have been used for comparison purposes.

Male Wistar rat albino with an average weight of 250 to 350 g were used for the test. The rats were stabilized for a period of ten days at a temperature of 21-1 ° C with controlled food intake and free water intake. Before the test, the animals were divided into groups of ten animals in order to achieve a good random distribution. Each dose was tested on three groups of animals. The animals were anesthetized with a dose of 30 mg / kg pentobarbital before the test. After the animals are under deep anesthesia *. the following substances were found on parenteral

Routes administered: i 1, the control animals: 1 ml / kg physiological * - 6 -

Saline, 2. the test animals: a solution of the substance to be tested in physiological saline with 1 ml / kg in the dosages given below in the table.

After 15 minutes, 16 If / kg of adrenaline were given intravenously in physiological saline to determine the protective effect of the substances in order to cause acute edema.

After five minutes the animals were sacrificed by decapitation, the lungs removed and their weight determined. The results of the experiment are summarized in Table 1, along with the concentration of the substances and the relative edema inhibition in percent. The results are discussed at the end of the tables.

TABLE 1 LINKAGE DOSE *% edema inhibition

Physiological solution _ n nn lml / kg + adrenaline * * υυ \

Aspirin 1 32.2 * 2.5 36.9 ". 5, 48.2" 10 54.3 '* 20 19.6 \ * aprotinin ** 30 17.3 100, 3Ö.5 "300 54.5 * · 1000 42.7

CONNECTION DOSIS *% LOCKING INHIBITION

- 7 - L-A.M. *** 1 21.2 2.5 27.4 5 29.0 "10 55.7 • 20 15.7 DA.M. **** 1 16.8 M 2.5 11.7 5 '15.4 10 4.4" 20 6.4 LA.E. ***** 0.1 3.4 0.25 32.2 " 0.5 33.3 1 55.7 "2.5 47.9" 5 41.0 "10 38.9 ·· 20 39.7 DA.E. ****** 0.1 7« 0.25 11 0.5 3 "1.0 4.2". 2.5 7 «H 5.0. 5.3 * 10 6 "20 2.5 L-Arg.lnin. 2HC1 1 7 ·· 2.5 9 5 · 11" 10 10.1 N ΟΛ 1 1

CONNECTION DOSIS% LOCKING INHIBITION

- 8 - ethanol ° · 08 7 «0.8 12 m 2 15 ·> 4 14.7.

8 1 "'m 16 15.9

Methanol ° * 00 2.3 «0.8 5 2 12« 4 6 8 15 «16 15 L-arginine. .2HC1 + ethanol 1 + 0.08 1 ° »2.5 + 0.8 9». 5 + 2 5 * 3 U 10 + 4 7 * 2 h 20 + 8 4 * 6 L-Arginino · 2HC1 + methanol 1 + 0.08 11.4 «2.5 + 0.8 7.2. * · «’ 5 + 2, 9 * 5 «10 + 4 8 · 1 * 20 + 8 6 * ln mg / kg, except aprotinin: the numbers mean * * U.I.K, / kgf i.e. Inhibitor units according to Kunitz ** the commercial product was used, which is sold by the Bayer paint factory under the name "Trasylol".

- 9 - *** L-A.M. = L-arginine ethyl ester hydrochloride.

D-A.M. = D-arginine methyl ester hydrochloride.

L-A.E. = L-arginine ethyl ester hydrochloride.

D-A.E. = D-arginine ethyl ester hydrochloride.

Protection against shock caused by endotoxins

The shock caused by endotoxin was caused by the method # described by A. Bertelli and L. Donati in the publication "The Influence of Some Enzymes and Enzymes Inhibitors in Shock" ("SHOCK Biochemical, Pharmaceutical and Clinical Aspects"; A. Bertelli and N. Back editor, Advances in Experimental Medicine and Biology; Vol 9, Plenum Press New York - London 1970, page 215).

For comparison, a substance with well-known therapeutic activity was used, in particular aprotinin, which is sold by the Bayer color factory under the trade name "Trasylol". Male Wistar rats weighing 120-10 g were used. The rats were previously stabilized for a period of ten days at a temperature of 21-1 ° C with controlled food intake with free water intake. Twenty-four hours before the test, the animals were divided into groups of ten animals in order to achieve a good random distribution. Each individual dose was examined in six groups of animals.

Lipopolysaccharide B from S. Enteritides (Difco Labs.) Was used as the endotoxin. This substance was administered at a dose - of 10 mg / kg endoperitoneally, one hour after the

Administration of the substance that was administered parenterally or in the form of a physiological solution with the same volume per kilogram to the animals used for comparison.

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The mortality in percent was determined on the test animals compared to the control animals after twenty-four hours. The results are summarized in Table 2.

Table 2

LINK DOSIS% MORTALITY

70.9

Physiological solution

Aprotinin 1000 50.0 »3000 65.0« 10000 65.0 «100000 933 L-A.M. 5 5.5.0 «io 50.0» 20 40.0 i50 50 28.0 D-A.M. 5 71.2 «10 68.5« 20 69 »50 73 L-A.E. 5 51.3 * n 10 48.2 h 20 37.5 h 50 25.0 D-A.E. 5 72 ». 10 67 .. 20 69.3 * »50 71 L-arginine .2HC1 5 69" 10 73 »20 71

LINK DOSIS% MORTALITY

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Methanol 2 76 .. 4 72 »8 7 4 ethanol 2 ^ 7 h 4 69 II 8 72 L-arginine.2HCl + methanol '4 + 0.61 73« 8 + 1.23 71 «' 16 + 2.46 75 L-arginine. , .2HCl + ethanol 4 + 1.06 69 "8 + 2.12 71" 16 + 4.24 74

The abbreviations are explained in Table 1. Here too, the doses for aprotinin are expressed in U.I.K./kg.

Protection against anaphylactic shock

The anaphylactic shock was caused by the method of S.M. Feimberg, J. Pharmaeol, Exp. Therap., 99.1 195 (1950).

It worked with male guinea pig albino with an average weight of 350 - 20 g / which were previously stabilized for ten days at a temperature of 21 - 1 ° C with controlled food intake with free water intake. Three days before the start of the experiment, the animals were divided into groups of ten animals / in order to achieve a good random distribution. Each single dose has been studied in three groups of animals.

Sensitization was by intravenous administration of "- 12-1 ml / guinea pig of horse serum (whole serum, not treated with preservative) diluted 1:10 with physiological saline. After 14 days, the solutions of the substances to be tested and the samples of physiological solutions for the confronters were administered parenterally. At the same time, horse serum, undiluted, (1 ml / guinea pig) was administered intravenously; this substance served as an agent for triggering anaphylaxis.

. The mortality in percent was determined after 12 hours.

The results are summarized in Table 3.

Table 3

LINK DOSIS% MORTALITY

Physiological solution “80

Aprotinin 3000 40 "10000 55” 100000 80 rL-A.M. 10 * 40.5 ** '20 27 "50 12.5 D-A.M. 10 78.8 ‘’ 20 81.0 *. .50 76.3

St "L-A.E. 10 35 M 20 23 "50 9.5»

LINK DOSIS% MORTALITY

- 13 - Λ in 76.7 D-A.E. 10 "20 79.4". 50 81.0 L-arginine .2HC1 10 78.5 w 20 ’76.3 50 80

Methanol 2 76.6, i »4 79.4« 8 81 J ethanol 2 76 · * 4 74 n 8, 76.5, L-arginine .2HCl + methanol 4 + 0.61 77.3 »8 + 1.23 76 · 3» «16 + 2.46 81 · ° L Arginine. .2HCl + ethanol 4 + 1.06 75 * 8 «· 8 + 2.12 73.2« 16 + 4.24 77 * ° »

For dosage and abbreviations see the signature for Table 1.

Inhibition of hyperfibrinolysis

The hyperfibrinolytic state is brought about by administration of urokinase. To determine the »* - 14 -

The F.D.P. (Fibrin Degradation Products) value is inhibited according to the procedure of J. Hawiger et al, J. Lab. Clin. Med. 21/93 (1970) using the staphylococcal agglutination test (Boehringer Mannheim GmbH).

Wister rats weighing 300-10 g / which were stabilized for a period of ten days at a temperature * 4 * * of -21 - 1 C with controlled food intake and free water intake serve as test animals. Twenty-four hours before the start of the experiment, the animals were divided into groups of ten. Divided animals to achieve a good random distribution. Each single dose was tested on three groups of animals,

At the start of the experiment, at time zero, the compounds to be tested were administered parenterally. An equal volume of physiological saline was administered to the confronters at the same time, except that some blood was drawn by cardiac puncture in order to determine the basic value of the fibrinolytic activity. After 30 minutes, an amount of 2500 U.I / rat human urokinase (product of Ukidan-Serono) was administered intravenously. After a further ten minutes, blood was withdrawn by heart puncture for the final determination of the F.D.P., a determination expressed in milliliters. The results obtained in this way are summarized in Table 4.

Table 4 - * LINK DOSIS F.D.P.

i. "- 0.26 basic value * Physiological solution - 3.30

Aprotinin 1000 0.34 «3000 0.29« 10000 0.30 Ή -15- CONNECTION_DOSIS_F.D.P.

Tranexamic Acid 5 0.58 »10 0.39 h 20 0.33 L-A.M. 5 0.48 "10 0.30" 20 0.25 Λ D-A.M. '5 3.10. Μ 10 3.03 "20 3.16 L-A.E. 5 0.50" 10 0.33 Μ 20 0.27 D-A.E. 5 3.20 * 10 3.16 * · 20 3.19 L-arginine .2HC1 5 3.28 "10 2.96" 20 3.15

Methanol · 2 3.02 "4 3.12 8 3.27 Ethanol 2 3.06" 4 2.95 "8 3.11 * · ui L-arginine .2HCl + methanol 4 + 0 ^ 61 3.00 v" 8 + 1.23 3.12 "16 + 2.46 3.20 L-arginine .2HC1 + Ethanol 4 + 1.06 2.98 «- 16 -

For dosage and abbreviations see the signature for Table 1.

Tranexamic acid is trans-4- (aminomethyl) cyclohexane-carboxylic acid.

The values reported in Tables 1 to 4 show the remarkable therapeutic effect of arginine ethyl 1 or methyl ester on the test dosage and the specificity of the activity with regard to the structure and also the stereoisomerism of the compound. In contrast, ‘unspecific effects and effects associated with those on the

Experimental animals, which were carried as control animals with physiological saline, were comparable if the animals were given arginine alone or the alcohols alone or mixtures of arginine and the alcohols or the D-arginine esters.

The toxicity of the L-arginine esters, the therapeutic activity of which has been illustrated in the examples above, was investigated in two animal species: intravenously in mice and intravenously and endoperitoneally in rats. The results at DL-50 mg / kg were:

Type: male Swiss mouse albums with an average + average weight of 22 - 1 h «

Route of administration: intravenous DL-50: 315.6 mg / kg (L.F. = 276.5 - 360.2)

Species: male Wistar rat alhinos with an average weight of 180 - 10 g route of administration: intravenous DL-50: 431.73 mg / kg (L.F. = 374.2 - 498.0)

Route of administration: endoperitoneal 'DL-50: 1111.1 mg / kg (LF = 1002.9 - 1230.8) 4 «- 17 - <* ï' Note that the toxicity values are in a range which is considerably higher is the range of doses used for therapeutic purposes.

At the dosages given above and in the manner of administration given above, the administration of the substances did not cause mortality in healthy animals and did not cause any visible signs of intoxication. The values compiled in Tables 1 to 4 show the therapeutic fourth of the pharmaceutical approach according to the invention.

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Claims (4)

1. Pharmaceutical approach for use as protection against acute pulmonary edema, anaphylactic shock and other types of schack as well as hyperfibrinolysis caused by agents which activate plasminogen, characterized by a solution of an ester of L-arginine with an aliphatic alcohol, or a ^ its salts with a pharmaceutically suitable organic or inorganic acid in lilasser at a pH between 5.5 and B, 5. 2. Approach according to claim 1 in single trials with 0.25 to 100 mg / kg body weight of the living being to be treated parenterally. 3. Approach according to claim 1, characterized in that the peat is caused by bacterial endotoxins. k. Batch according to claim 1, characterized in that the said ester of L-arginine is present as a hydrochloric salt. 5. Approach according to claim 1, characterized in that the alcohol is methyl or ethyl alcohol. s \. 'Λ